Multi-Stimuli-Responsive Microcapsules for Adjustable

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https://www.sodocs.net/doc/5611360862.html, pH-responsive, [13–27]magnetic-respon-sive, [28–37] and other stimuli-responsive microcapsules in recent years. However,

in many cases, different environmental changes may occur at the same time, thus single stimulus-responsive micro-capsules are insuf? cient for practical applications. Therefore, it is much more favorable that microcapsules possess

multiple stimuli-responsive properties simultaneously. [38–41] More importantly, the patients’ conditions are usually com-plex and diverse. To achieve best effects

and reduce side effects of drugs, it is necessary to regulate the release dosage and the release rate in time according

to patients’ individual differences. How-ever, the design and preparation of multi-stimuli-responsive microcapsules with

adjustable controlled-release have not been reported yet.

U p to now, the stimuli-responsive microcapsules used as drug delivery systems are mainly devel-oped based on the “on-off” mechanism, which can be divided

into two categories. The ? rst one is prepared by grafting stimuli-responsive materials into the pores of microcap-sules, [1,2,5,11,12,37,38,42] or incorporating stimuli-responsive parti-cles into the microcapsule membranes. [4,43] The microcapsule

substrates have no environmental response and the controlled-release properties only depend on the abrupt deswelling/swelling properties of the graft chains or the embedded par-ticles, which can regulate the trans-membrane diffusional permeation of drug molecules. Another one is fabricated by

directly utilizing the stimuli-responsive materials as the micro-capsule membrane. The release mechanisms of these micro-capsules mainly rely on the deswelling/swelling properties of

the capsule membrane themselves,

[7–10,13–15,19,22–24,26,28,35,39–41] or the squeezing action from sudden shrinking of the capsule

membranes, [36,44,45] or the decomposition of capsule mem-branes. [16,17,20,46,47] Generally, due to the “on-off” state of micro-capsules, the drug controlled-release is often in an extreme “on ” or “off” state. Their drug release rate can not be ? exibly adjusted according to patients’ individual differences; there-fore, smart microcapsules with adjustable controlled-release rate are more rational for drug administration. Besides, pH is an important stimulus for stimuli-responsive controlled-release systems, because pH differentiation exists at many speci? c and pathological sites in human body. Temperature is also an important factor that can be easily controlled, and the site-speci? c targeting drug delivery can be achieved easily by M

ulti-Stimuli-Responsive Microcapsules for Adjustable Controlled-Release

J ie W ei ,X iao-Jie J u ,*X iao-Yi Z ou ,R ui X ie ,W ei W ang ,Y ing-Mei L iu ,a nd L iang-Yin C hu * N ovel multi-stimuli-responsive microcapsules with adjustable controlled-release characteristics are prepared by a micro? uidic technique. The proposed microcapsules are composed of crosslinked chitosan acting as

pH-responsive capsule membrane, embedded magnetic nanoparticles to

realize “site-speci? c targeting”, and embedded temperature-responsive sub-microspheres serving as “micro-valves”. By applying an external magnetic ? eld, the prepared smart microcapsules can achieve targeting aggregation at speci? c sites. Due to acid-induced swelling of the capsule membranes, the

microcapsules exhibit higher release rate at speci? c acidic sites compared to

that at normal sites with physiological pH. More importantly, through control-ling the hydrodynamic size of sub-microsphere “micro-valves” by regulating the environment temperature, the release rate of drug molecules from the microcapsules can be ? exibly adjusted. This kind of multi-stimuli-responsive microcapsules with site-speci? c targeting and adjustable controlled-release

characteristics provides a new mode for designing “intelligent” controlled-release systems and is expected to realize more rational drug administration.

DOI: 10.1002/adfm.201303844

J . Wei, Prof. X.-J. Ju, X.-Y . Zou, Prof. R. Xie, Dr. W. Wang, Y .-M. Liu, Prof. L.-Y . Chu

S chool of Chemical Engineering Sichuan University C hengdu ,S ichuan 610065 ,P . R. China E-mail: c huly@https://www.sodocs.net/doc/5611360862.html,; j uxiaojie@https://www.sodocs.net/doc/5611360862.html,

P rof. L.-Y . Chu

S tate Key Laboratory of Polymer Materials Engineering and Collaborative Innovation Center for Biomaterials Science and Technology Sichuan University

C hengdu ,S ichuan 610065 ,P . R. China 1. I ntroduction

S mart microcapsules, which can control the release of their encapsulated contents according to various environmental stimuli, have attracted great interests from various ? elds in recent years. Because of their relatively faster response rate and other advantages such as small size, large inner volume, huge total surface area and stable capsule membrane, these environmental stimuli-responsive microcapsules are consid-ered to be the most ideally intelligent drug delivery systems. By encapsulated inside these microcapsules, drugs or chemi-cals can be released at a desired rate only when and/or where the release is needed. [1] A considerable amount of researches have been carried out on temperature-responsive, [2–12]

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? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim magnetic targeting. Consequently, the design and preparation of microcapsules with pH-, temperature- and magnetic-respon-sive synergistic effects and adjustable controlled-release rate are of great potential in drug delivery.

I n this study, we report on a novel multi-stimuli-responsive microcapsule with adjustable controlled-release by embedding temperature-responsive sub-microspheres as “micro-valves” into the magnetic- and pH-responsive microcapsule mem-brane. The proposed microcapsules can simultaneously achieve targeted delivery by applying an external magnetic ? eld, self-regulated drug release according to pH difference at patholog-ical sites, and adjustable controlled-release relying on temper-ature regulation. That is, this kind of novel microcapsule can achieve targeting aggregation at speci? c pathological sites and effectively adjustable controlled-release according to patients’ individual differences, which is of great importance for real-izing more rational drug administration.

2. R esults and Discussion

2.1. S trategy for Preparation of Microcapsules

T

he fabrication procedure and the controlled-release mech-anism of the proposed microcapsules are schematically illustrated in F igure 1 . The microcapsule is composed of bio-compatible and pH-responsive chitosan capsule membrane embedded with magnetic nanoparticles and temperature-responsive sub-microspheres. For drug delivery systems, micro-capsules with narrow size distribution are preferable, since the drug loading levels and release kinetics are directly affected by the size distribution of microcapsules. Micro? uidic techniques, which have been developed for generating highly monodis-perse emulsions in recent years,

[ 48,49 ] are used for preparing microcapsules with uniform size. As shown in Figure 1 A , the chitosan aqueous solution containing magnetic-responsive

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, 3312–3323 F igure 1. S chematic illustration of fabrication process (A–D ) and controlled-release mechanism (E–G) of the proposed multi-stimuli-responsive microcapsules with adjustable controlled-release. The capillary micro? uidic device (A) is used for generating O/W/O double emulsions (B), and microcapsules are prepared by using these emulsions as templates via crosslinking reaction (C,D). When pH > p K a , the microcapsule is in a shrunken state, therefore the release rate is low (E); while a high release rate is resulted due to the swollen state of the microcapsule when pH < p K a (F). By increasing environmental temperature, the interspace size between the capsule membrane and sub-microspheres becomes larger due to the shrinkage of sub-microspheres, so the drug release rate is further increased (G). The pH-/thermo-responsive behaviors are reversible.

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https://www.sodocs.net/doc/5611360862.html, ? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim nanoparticles and temperature-responsive sub-microspheres is used as the middle ? uid (MF), while the oil phase containing crosslinker glutaraldehyde (GA) is used as the inner ? uid (IF) and outer ? uid (OF). The obtained oil-in-water-in-oil (O/W /O) emulsions (Figure 1 B ) are used as templates for the prepara-tion of multi-stimuli-responsive microcapsules (Figure 1 C ,D).

B y embedding magnetic nanoparticles into the microcap-sule membrane, site-speci? c targeting can be achieved under an external magnetic ? eld. Crosslinked chitosan have typical cationic pH-responsive properties. At speci? c sites where the environmental pH is higher than the p K a value of chitosan

(about 6.2–7.0), [ 16 ] such as in the normal tissue (the body physi-ological pH is about 7.4), the microcapsule membrane is in

the compact state due to the pH-responsive shrinking, which results in a low release rate of drugs from the microcapsule (Figure 1 E ). On the other hand, when the environmental pH is lower than the p K a value of chitosan, such as at some chronic

wound sites whose pH values are as low as 5.45,

[ 50 ] the micro-capsule membrane is in the loose state due to the pH-responsive swelling, which leads to a high release rate (Figure 1 F ). That is, the prepared smart microcapsules can realize self-regulated drug release according to pH difference at pathological sites. More importantly, through the addition of temperature-respon-sive sub-microspheres as “micro-valves”, drug release rate can be effectively adjusted through regulating external tempera-ture via local heating/cooling. The interspace size between the microcapsule membrane and sub-microspheres can be ? exibly regulated through the temperature-dependent volume change of sub-microspheres, which results in adjustable controlled-release properties. When ambient temperature is increased, the interspaces become lager due to the shrinking of sub-micro-spheres, and therefore drug release rate is higher (Figure 1 G ); on the contrary, when the temperature is decreased, the inter-spaces become smaller due to the swelling of sub-microspheres, and then a lower release rate is resulted (Figure 1 F ).

2.2. C haracterization of Magnetic Nanoparticles and Tempera-ture-Responsive Sub-Microspheres

T he morphology of the magnetic nanoparticles is observed by transmission electron microscopy (TEM). As shown in F igure 2 A , the size of prepared magnetic nanoparticles is around 20 nm and there is no obvious aggregation, which indi-cates that the nanoparticles are well dispersed in water. Mag-netic property of the nanoparticles is measured with a vibrating

sample magnetometer (VSM). As shown in Figure 2 B , the saturation magnetization ( M s ) of the magnetic nanoparticles is 72.32 emu g ?1 , and hysteresis and coercivity are almost unde-tectable, which suggests that the superparamagnetic property of the prepared nanoparticles is satisfactory.

T he temperature-responsive sub-microspheres are labeled with red ? uorescence dye to make them easy to be observed. Moreover, in order to make the temperature-responsive sub-microspheres have obvious volume phase transition near human body temperature (37 °C), acrylamide (AAm) is used as a hydrophilic comonomer to modulate the thermo-sen-sitivity of the sub-microspheres. Figure 2 C shows the con-focal laser scanning microscope (CL SM) image of obtained poly( N -isopropylacrylamide- c o -acrylamide) (P(NIPAM- c o -AAm))

sub-microspheres in water at room temperature on the red ? uo-rescent channel excited at 543 nm. The sub-microspheres show red ? uorescence and exhibit good monodispersity, and are well-dispersed in water. The equilibrium deswelling ratio ( D T /D 25°C )of P(NIPAM- c o -AAm) sub-microspheres as a function of tem-perature is shown in Figure 2 D . The results show that these temperature-responsive sub-microspheres deswell gradually with increasing temperature and the equilibrium deswelling ratio shows signi? cant change near body temperature (37 °C). This kind of temperature-dependent volume phase transition provides feasibility for subsequent adjustable controlled-release near body temperature.

2.3. M orphologies of Emulsions and Microcapsules

T he optical and ? uorescent microscope images of O/W /O double emulsions generated by the micro? uidic method are shown in F igure 3 . The emulsions, which act as templates for fabricating microcapsules via crosslinking reaction, are highly monodisperse and quite stable (Figure 3 A ′–C ′). Because only temperature-responsive sub-microspheres are labeled with red ? uorescence, emulsions with only chitosan in middle water phase (Figure 3 A ) or with both chitosan and magnetic nanoparticles in middle water phase (Figure 3 B ) exhibit no ? uorescence; while emul-sions containing chitosan, magnetic nanoparticles and sub-microspheres (Figure 3 C ) show clearly red ? uorescence. F igure 4 shows CLSM images and optical microscope images of different microcapsules crosslinked from different double emulsion templates. The microcapsules are dispersed in buffer solution of pH 7.4 at 37 °C, which simulate the body physiolog-ical pH and temperature. Under the same condition, only chi-tosan microcapsules containing both magnetic nanoparticles and temperature-responsive sub-microspheres (CS-M-T, Figure 4 C ) show red ? uorescent, while the chitosan microcapsules (CS, Figure 4 A ) and the magnetic nanoparticles embedded chitosan microcapsules (CS-M, Figure 4 B ) show no ? uorescence. The red ? uorescence from the capsule membranes of CS-M-T micro-capsules indicates the successfully embedding of temperature-responsive sub-microspheres into the microcapsules. The optical microscope images of different microcapsules (Figure

4 A ′–C ′) look almost the same, which all show that the obtained micro-capsules are with good sphericity and monodispersity. 2.4. I n?

uences of Preparation Conditions on pH-Sensitivities of Microcapsules

M icrocapsules with pH-sensitivity are studied widely, because pH difference exists at many physiological, biological and/or chemical systems. Chronic wounds have been reported to have pH values between 8.65 and 5.45,

[ 50 ] and cancer tissue is also reported to be acidic extracellularly.

[ 51,52 ] The cationic pH-responsive microcapsules, which have acid-responsive swelling property due to protonation at acidic conditions, are suitable for rate-controlled release and sustained drug release in acidic con-ditions via self-regulated adjustment of molecular diffusion per-meation. Chitosan is a well-known cationic polysaccharide with

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? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim excellent biological activity, good biocompatibility and biodegra-dability, so microcapsules based on chitosan are attracting more and more interests in various applications. Microcapsules made from crosslinked chitosan exhibit typical cationic pH-respon-sive characteristics. When the environmental pH is lower than the p K a value of chitosan, the microcapsules swell due to the protonation of amino groups, and when the pH is higher than its p K a value, the deprotonation of amino groups results the deswell of microcapsules. Swelling ratios ( O D pH /O D p H7.4)of microcapsule outer diameter at certain pH to that at body physiological pH 7.4 are used to characterize the acid-induced swelling change of these prepared microcapsules. T he in? uences of the preparation conditions on the pH-sensitivities of microcapsules are investigated and the recipes of different ? uids are listed in T able 1 . As shown in F igure 5 , all microcapsules exhibit good pH-sensitivity, and the swelling ratios increase with decrease of external pH values. From Figure 5 A it can be seen that, with the same IF and MF, the chitosan microcapsules prepared with different GA concen-trations in OF show different swelling extents. The chitosan microcapsules prepared with lower GA concentration exhibit higher swelling ratio compared with that prepared with higher GA concentration. The similar results are also observed from microcapsules prepared with different GA concentrations in IF, as shown in Figure 5 B . GA is the crosslinker for chitosan microcapsules. With the decrease of GA concentration, the crosslinking density of microcapsules decreases, which results the increase in swelling ratios of microcapsules. Microcap-sules prepared with too low crosslinking density are easy to be broken, so GA concentrations of IF and OF are chosen to be 1/50 (v/v) in the subsequent studies.

F or the preparation of chitosan microcapsules, hydroxyethyl cellulose (HEC) is added into MF to increase ? uid viscosity. The chitosan microcapsules prepared with HEC addition are much stable than those prepared without HEC addition. The pH-responsive behaviors of microcapsules prepared with and without addition of HEC are also studied. Microcapsules pre-pared without HEC addition show slightly larger swelling ratio than that prepared with HEC addition at a same pH condi-tion (Figure 5 C ). The reason is that HEC is a macromolecular compound, which has long polymer chains. The entangle-ments between HEC and chitosan molecular chains restrict the swelling of microcapsules, so the lower swelling ratio of micro-capsules prepared with HEC addition is resulted. Considering

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, 3312–3323 F igure 2. A ) TEM image of the prepared magnetic nanoparticles, B) magnetization curve of magnetic nanoparticles at room temperature, C) CLSM image of the temperature-responsive sub-microspheres in water at room temperature on red ? uorescent channel, and D) temperature-responsive property of sub-microspheres.

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B y adding magnetic nanoparticles into MF, chitosan micro-capsules with magnetic-sensitivity (CS-M) are obtained. With the addition of magnetic nanoparticles and thermo-sensitive sub-microspheres into MF, the generated chitosan microcap-sules possess both magnetic-sensitivity and thermo-sensitivity (CS-M-T). The pH-sensitivities of these microcapsules are also investigated, as shown in Figure 5 D . The addition of magnetic nanoparticles have almost no in? uence on the pH-sensitivity of

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F igure 3. A –C) CLSM images of different O/W/O double emulsion templates on red ? uorescent channel; and A ′–C ′) optical microscope images of dif-ferent emulsion templates. (A,A’) represent emulsions prepared with only chitosan in MF (4#), (B,B ′) represent emulsions prepared with both chitosan and magnetic nanoparticles in MF (5#), C and C’ represent emulsions prepared with chitosan, magnetic nanoparticles and temperature-responsive

sub-microspheres in MF (6#). Scale bars are all 500 μm.

F igure 4. A –C) CLSM images of different microcapsules on red ? uorescent channel; and A ′–C ′) optical microscope images of different microcapsules.

(A,A ′) represent CS microcapsules, (B,B ′) represent CS-M microcapsules, (C,C ′) represent CS-M-T microcapsules. The microcapsules are dispersed in buffer solution of pH 7.4 at 37 °C. Scale bars are all 250 μm.

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? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim chitosan microcapsules, but the addition of sub-microspheres

have slight in? uence on the pH-sensitivity of microcapsules. The magnetic nanoparticles are very small (about 20 nm) and the amount of nanoparticles added into the microcapsules is relatively low (0.15% w/v), so the pH-sensitivity of micro-capsules is nearly not in? uenced by the addition of magnetic nanoparticles. However, the temperature-responsive sub-micro-spheres are relatively large (about 1 μm in water at room tem-perature) and the content of sub-microspheres in the micro-capsules is relatively high (1%w/v). So, CS-M-T microcapsules exhibit slightly lower pH-responsive swelling ratios than CS and CS-M microcapsules at the same condition.

2.5. M agnetic Properties of Microcapsules

D rug release at speci? c pathological sites can effectively reduce

the side effects. Incorporation of superparamagnetic nanopar-ticles into the chitosan capsule membrane enables the micro-capsules to realize magnetic-guided targeting delivery. The CS-M ( F igure 6A ,A ′,A ″) and CS-M-T (Figure 6 B ,B ′,B ″) micro-capsules both exhibit satisfactory magnetic properties. Without the external magnetic ? eld, the microcapsules are randomly dispersed in water (Figure 6 A ,B); while after applying the external magnetic ? eld, the microcapsules are aggregated at the sites where the magnet is placed (Figure 6 A ′,B ′). A VSM is also

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, 3312–3323 F igure 5. p H-responsive swelling ratios of CS microcapsules prepared A) with different concentrations of GA in OF, B) with different concentrations

of GA in IF, C) with and without HEC addition in MF, and D) pH-responsive swelling ratios of CS, CS-M, and CS-M-T microcapsules. T = 37 °C.

T able 1. T he recipes of different ? uids.

Code IF (Inner Fluid)

MF (Middle Fluid)

OF (Outer Fluid)

1#Oil( V G A-S-BB :V SO = 1:20) +3%w/v PGPR Pure water+4%w/v CS+0.25%w/v HEC+0.5%w/v F127Oil( V GA-S-BB :V SO = 1:50) +8%w/v PGPR 2#Oil( V G A-S-BB :V SO = 1:20) +3%w/v PGPR Pure water+4%w/v CS+0.25%w/v HEC+0.5%w/v F127Oil( V GA-S-BB :V SO = 1:75) +8%w/v PGPR 3#Oil( V GA-S-BB :V S O = 1:50) +3%w/v PGPR Pure water+4%w/v CS+0.25%w/v HEC+0.5%w/v F127

Oil( V G A-S-BB :V S O = 1:50) +8%w/v PGPR 4#Oil( V G A-S-BB :V S O = 1:50) +3%w/v PGPR Pure water+4%w/v CS+0.5%w/v F127

Oil( V G A-S-BB :V S O = 1:50) +8%w/v PGPR 5#Oil( V GA-S-BB :V S O = 1:50) +3%w/v PGPR Pure water+4%w/v CS+0.5%w/v F127+0.15%w/v magnetic nanoparticles

Oil( V G A-S-BB :V S O = 1:50) +8%w/v PGPR 6#

Oil( V GA-S-BB :V SO = 1:50) +3%w/v PGPR

Pure water+4%w/v CS+0.5%w/v F127+0.15%w/v magnetic

nanoparticles+1%w/v sub-microspheres

Oil( V G A-S-BB :V SO = 1:50) +8%w/v PGPR

(Note: “ V G A-S-BB ” represents the volume of glutaraldehyde-saturated benzyl benzoate; “ V S O

” represents the volume of soybean oil.)

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https://www.sodocs.net/doc/5611360862.html, ? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim used to measure the magnetic properties of CS-M and CS-M-T microcapsules and their magnetization curves are displayed in Figure 6A ″ and Figure 6 B ″, respectively. Their hysteresis and coercivity are almost undetectable, which suggests that the CS-M and CS-M-T microcapsules also have superparamagnetic properties. The M s of CS-M and CS-M-T microcapsules are

4.47 emu g ?1 and 4.11 emu g ?1 respectively, which are much

lower than that of pure magnetic nanoparticles (72.32 emu g ?1).

This is attributed to the low content of nanoparticles in the microcapsule membrane. To con? rm the nanoparticles can be stably kept inside the capsule membranes, the magnetic prop-erties of nanoparticle-embedded microcapsules before and after repeatedly swelling/shrinking for 20 times are compared. The results show that M s values of CS-M-T microcapsules before and after repeatedly swelling/shrinking for 20 times are almost the same (Figure 6 B ″), which indicates that the nanoparticles embedded in the capsule membranes do not diffuse out even when the membranes undergo repeated swelling. Before being added in the chitosan solution, the nanoparticles are negatively charged because they have been modi? ed with trisodium cit-rate. While the chitosan is positively charged at acidic condi-tions (pH < p K a of chitosan). Although the capsule membranes are swollen in the acidic conditions, the nanoparticles can be kept stably inside the membrane due to electrostatic attraction between the negatively charged nanoparticles and the positively charged chitosan networks.

2.6. C ontrolled-Release Characteristics of Multi-Stimuli-Respon-sive Microcapsules

T

he pH-responsive controlled-release behaviors of Vitamin B12 (VB12) as model drug from CS-M-T microcapsules are shown in F igure 7 . The permeability coef? cient of VB12 molecules ( P VB12

) from microcapsules exhibits obvious pH-dependent characteristics. When the environmental pH is lower than the body physiological value (pH 7.4), the value of P VB12increases with decreasing the pH value in external circumstances. The capsule membrane is in the loose state in acidic conditions due to the microcapsule swelling and then a high release rate is resulted. Another parameter de? ned as P pH /P p H7.4 is also intro-duced to characterize the permeability change degree compared to that at the body physiological pH. It can be seen that the P pH /P p H7.4 value also increases with decreasing pH value. As expected, the pH-responsive controlled-release behaviors of the CS-M-T microcapsules are consistent with their pH-dependent swelling results.

T he adjustable drug controlled-release of microcapsules is achieved by regulating the interspace size between the micro-capsule membrane and sub-microspheres, while the interspace size is determined by the size change of temperature-respon-sive sub-microspheres embedded in the capsule membrane. To estimate the adjustable controlled-release behaviors induced by temperature change, the pH value of external circumstances

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F igure 6. M agnetic-responsive property of CS-M and CS-M-T microcapsules. Microcapsules in buffer solution of pH 7.4 at room temperature A,B) without magnet and A ′,B ′)with an external magnet, and A",B") magnetization curves of microcapsules at room temperature. (A,A ′,A ″) represent CS-M microcapsules, and (B,B ′,B ″) represent CS-M-T microcapsules.

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? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim should be ? xed. Very low colonic pH values have been found in some severe active ulcerative colitis (the lowest pH values are 2.3–3.4) and some chronic wounds have been reported to have pH value as low as 5.45. [ 50,53 ] Herein, to re? ect the adjustable

controlled-release under pathologically acidic conditions, two pH values (pH 3 and pH 5) are chosen in this work.

T he adjustable controlled-release of VB12 from CS-M-T microcapsules at pH 3 in response to temperature is exhib-ited in F igure 8 A ,B. The CS-M microcapsules are used as the control group. It is desirable that the P VB12 value of CS-M-T microcapsules increases with increasing of temperature from 25 °C to 49 °C (Figure 8 A ). In particular, the P VB12 value of CS-M-T microcapsules increases signi? cantly in the tempera-ture range of 34–43 °C, while it increases relatively slowly in the temperature ranges of 25–34 °C and 43–49 °C. The tem-perature-dependent P VB12 change of CS-M-T microcapsules corresponds well with the temperature-sensitivity of the sub-microspheres shown in Figure 2 D . The temperature-responsive sub-microspheres are used as the “micro-valves” to adjust the

release rate of microcapsules. Interspace size between the cap-sule membrane and sub-microspheres, which determines the VB12 release rate from the microcapsules, can be adjusted by temperature-dependent volume change of sub-microspheres. Therefore, the changing trend of VB12 permeability coef-? cients is consistent well with the temperature-responsive volume change of sub-microspheres. When the temperature is increased, the sub-microspheres embedded into the capsule membrane shrink, which causes the interspaces between the capsule membrane and sub-microspheres for VB12 passing through become larger. As a result, the value of P VB12becomes

higher with increasing temperature. While when the tempera-ture is decreased, the interspaces become smaller due to the swelling of sub-microspheres, therefore resulting in a lower value of P VB12 . In contrast, the permeability coef? cients of VB12 from CS-M microcapsules changes slightly in the whole temperature range (25–49 °C), and there is no obviously change in the range of 34–43 °C under the same condition. This slight change of P VB12 is due to the accelerated diffusivity of VB12

with increasing temperature. From practical application point of view, the adjustable controlled-release should be operated near body temperature (37 °C). A parameter de? ned as P T /P 37 °C is introduced to evaluate the adjustable controlled-release char-acteristics with respect to VB12 release near body temperature, in which P T is the permeability coef? cient of VB12 from micro-capsules at certain temperature T , while P 37 °C is the perme-ability coef? cient of VB12 at 37 °C. From Figure 8 B it can be seen that the CS-M-T microcapsules can achieve a better and more effective adjustment of drug release in the temperature range near 37 °C than CS-M microcapsules through controlling the temperature due to the embedded temperature-responsive sub-microspheres.

T he adjustable release characteristics of VB12 at pH 5 are also studied, as shown in Figure 8 C ,D. Just like the release behaviors at pH 3, the permeability coef? cient of VB12 from CS-M-T microcapsules increases signi? cantly with the increase of temperature in the range of 34–40 °C, while the P VB12of CS-M microcapsules changes slightly under the same circum-stances. P T /P 37 °C, as a function of temperature, also re?

ects the effective adjustment of drug controlled-release for CS-M-T

microcapsules at pH 5 in the temperature range near 37 °C. That is, the CS-M-T microcapsules can also achieve an effective adjustable drug delivery than CS-M microcapsules at pH 5.

T he temperature-controlled release behaviors of microcap-sules at normal body physiological pH (about 7.4) are also

studied. At pH 7.4, which is higher than the p K a value of chi-tosan, the chitosan membranes are in the compacted state. Here, FITC-dextran is selected as the model drug. The per-meability of the capsule membrane can also be modulated by changing the temperature even when chitosan is in the com-pacted state at pH 7.4 (

F igure 9 ). The release rate of FITC-dextran from CS-M-T microcapsules also increases signi?

-cantly with the increase of temperature. In contrast, there is no obvious change in the release rate of FITC-dextran from CS-M microcapsules when the temperature is varied in the same range. That is, even when chitosan membranes are in the com-pacted state at pH 7.4, the embedded temperature-responsive sub-microspheres are still ef? cient as “micro-valves”. T he in? uences of the added amount of sub-microspheres into the capsule membrane on the controlled-release behaviors are also investigated. The controlled-release characteristics of

VB12 from CS-M-T microcapsules containing different con-tents of sub-microspheres at temperature of 37 °C and pH of

3 are shown in F igure 10 . With increasing the added amount of sub-microspheres, the value of P VB12 increases obviously.

The number of interspaces between the capsule membrane and sub-microspheres is directly determined by the added amount of sub-microspheres. Consequently, with the increase of sub-microspheres amount, the value of P VB12 increases due to the increase of the number of interspaces for VB12 passing through. When the added amount of sub-microspheres is low, the sub-microspheres are well dispersed in the capsule mem-brane and thus they do not form percolated networks. However,

when a large amount of sub-microspheres are added in the cap-sule membrane, the sub-microspheres may be arrayed closely one by one although they are well dispersed, so that some con-nected porous networks may be formed through the shrinkage

of sub-microspheres at 37 °C. Therefore, when a large amount Adv. Funct. Mater. 2014, 24

, 3312–3323 F igure 7. p H-responsive release of model drug VB12 from CS-M-T micro-capsules at 37 °C.

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of sub-microspheres are added, the permeability of CS-M-T microcapsules at 37 °C increases a lot. A parameter de? ned as P C /P 0 is also used to re? ect the in? uence degree of the addition amount of sub-microspheres on the VB12 controlled-release, in which P C is the permeability coef? cient of VB12 from CS-M-T

microcapsules prepared with certain concentration C of sub-microspheres and P 0 is that from chitosan microcapsules pre-pared without sub-microspheres. With increasing the amount

of embedded sub-microspheres in the capsule membrane, the in? uence degree on the VB12 controlled-release also increases.

3. C onclusions

I n summary, by embedding magnetic nanoparticles and temperature-responsive sub-microspheres into the capsule membrane of a pH-responsive microcapsule, a novel multi-stimuli-responsive microcapsule with adjustable controlled-release characteristics is successfully prepared in this work. By introduction of hydrophilic AAm units, the temperature-responsive sub-microspheres working as “micro-valves” exhibit signi? cant volume phase transition near human body tempera-ture. Due to the superparamagnetic property of magnetic nano-particles, the prepared multi-stimuli-responsive microcapsules can achieve targeting aggregation at speci? c sites by applying an external magnetic ? eld. Furthermore, these smart microcap-sules exhibit cationic pH-responsive controlled-release proper-ties, which are well consistent with their acid-induce volume swelling behaviors. More importantly, the interspace size between the capsule membrane and sub-microspheres, which controls the VB12 release rate from the microcapsules, can be tuned through the temperature-responsive volume change

of the sub-microspheres. As a result, the smart microcapsules

F igure 8. A djustable controlled-release characteristics of model drug VB12 from CS-M and CS-M-T microcapsules in buffer solutions with different

pH values and at different temperatures.

F igure 9. R elease behaviors of FITC-dextran from CS-M and CS-M-T microcapsules in buffer solution of pH 7.4 at different temperatures. The ? uorescence intensity is measured in the region that covers the microcapsules.

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? 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Funct. Mater. 2014, 24, 3312–3323exhibit temperature-dependently adjustable controlled-release characteristics. Such multi-stimuli-responsive microcapsules are promising to achieve a more rational drug delivery and controlled-release according to patients’ individual differences.

4. E xperimental Section M aterials : Water-soluble chitosan ( M w = 5000, degree of deacetylation

= 85%) is purchased from Ji’nan Haidebei Marine Bioengineering Co., Ltd. N -isopropylacrylamide (NIPAM), purchased from Sigma-Aldrich, is puri? ed by recrystallization with a hexane/acetone mixture. GA,

HEC (Mw ≥ 300 000), AAm, N ,N ′-methylene-bis-acrylamide (MBA),

potassium persulfate (K 2S 2O 8 ), iron (III) chloride (FeCl 3·6H 2O ) and

iron (II) chloride (FeCl 2·4H 2

O ) are all purchased from Chengdu Kelong Chemical Reagents Co., Ltd. Pluronic F-127 (F127) and FITC-dextran ( M w = 4000) are purchased from Sigma-Aldrich. Benzyl benzoate is obtained from Sinopharm Chemical Reagent Co., Ltd. Soybean oil is provided by Kerry Oils & Grains Co., Ltd. Methacryloxy thiocarbonyl

rhodamine B (Poly? uor 570) is purchased from Polysciences. All other chemicals are of analytical grade and used as received. Pure water

(18.2 M Ω at 25 °C) from a Milli-Q Plus water puri? cation system (Millipore) is used throughout the experiments.

S ynthesis and Characterization of Magnetic-Responsive Nanoparticles and T emperature-Responsive Sub-Microspheres :Magnetic-responsive

nanoparticles are prepared by the chemical coprecipitation method as reported. [ 36,54 ]Brie? y, hydrochloric acid (4 mL) and FeCl 2·4H 2O (7.2 g) are dissolved into pure water (20 mL). The resultant solution is mixed with FeCl 3·6H 2

O aqueous solution (27 wt%, 29 mL). Afterward, pure water is added to make the aqueous solution up to 100 mL. By adding ammonia (40 mL), the solution turns to dark indicating that magnetite nanoparticles are synthesized. To make the nanoparticles well dispersed in water, the obtained nanoparticles are further modi? ed

with trisodium citrate. After adding nitric acid (14.4 mL) to acidi? cation,

a boiling solution of ferric nitrate (20 wt%, 50 mL) is added into the

above magnetite solution. After magnetic decantation, the precipitate is dispersed in a solution of trisodium citrate salt (14 wt%, 50 mL). The mixture is heated at 80 °C for half an hour and then the magnetic

nanoparticles are precipitated by addition of acetone. The precipitated nanoparticles are isolated and then suspended in pure water leading to a stable colloidal dispersion.

[ 55,56 ] The morphology of the prepared magnetic-responsive nanoparticles is observed by TEM (JEM-100CX, JEOL). The magnetic property of the nanoparticles is measured by a VSM (7400, Lakeshore) at room temperature.

T emperature-responsive sub-microspheres are prepared by precipitation polymerization.

[ 57,58 ] First, NIPAM (1.92 g), AAm (0.21 g), MBA (0.154 g), and a small amount of ? uorescence dye (Poly? uor 570) are dissolved in pure water (320 mL). Then, the solution is heated to 70 °C and bubbled with nitrogen gas to remove dissolved oxygen in the system. Approximately 10 min later, the K 2S 2

O 8 initiator solution (1.6 wt%, 5 ml) is added into the reactor to initiate the polymerization, and the reaction is maintained at 70 °C for 4 h under stirring. The resultant ? uorescence-labeled P(NIPAM- c o -AAm) sub-microspheres are puri? ed by centrifugation (Biofuge Primo R, Sorvall) to remove the unreacted monomers, crosslinker and initiator. The ? uorescence and dispersity of the sub-microspheres in water are observed at room temperature using a CLSM (SP5 II, Leica) and the red ? uorescent channel is excited at 543 nm. The hydrodynamic diameter of the sub-microspheres is measured by dynamic light scattering (D LS, Zetasizer Nano, ZEN 3690, Malvern). The temperature-sensitivity of P(NIPAM- c o -AAm) sub-microspheres is studied by estimating their size change at different temperatures in the range of 25 °C to 49 °C.

P reparation and Characterization of Microcapsules :Chitosan microcapsules (CS), chitosan microcapsules with magnetic-sensitivity (CS-M), and chitosan microcapsules with both magnetic- and thermo-sensitivity (CS-M-T) are all prepared using O/W/O double emulsions as crosslinking templates by a micro? uidic technique. The capillary micro? uidic device is

assembled according to previous work.

[ 48,49 ] The compositions of different ? uids are listed in Table 1 . The generated O/W/O emulsions are collected in a container, and then left to stand for 24 h to make sure the chitosan

in the water phase is completely crosslinked. The obtained microcapsules are washed using a mixture of ethyl acetate and isopropanol (1/5, v/v) to remove the inner and outer oil solutions, and ? nally the microcapsules are dispersed into water for further characterization. T he morphologies and ? uorescence of obtained emulsions and

microcapsules are observed using the CLSM. The pH-sensitivities of

different microcapsules are studied by evaluating their pH-responsive swelling behaviors in acidic conditions. Here, 0.01

M citric acid and 0.01

M disodium are used to adjust pH values of buffer solutions ranging from 3.0 to 7.4, and the ionic strength of pH buffer solutions is adjusted to 0.1 M . The microcapsules are immersed in a series of buffers for 24 h before measurement. The pH-dependent size changes of these microcapsules at 37 °C are measured according to their corresponding micrographs taken by an optical microscope (BX61, Olympus) equipped with a CCD camera and a thermostatic stage system (TS62, Instec). A VSM is also used to study the magnetic properties of microcapsules.

C ontrolled-Release Experiment : The controlled-release experiments of VB12 are carried out with a previous published method.

[ 1,11,12,37,42,59 ]

The prepared microcapsules are dialyzed against VB12 aqueous solution with a known concentration for more than three days to load VB12

inside the microcapsules. The dialysis is carried out at a certain pH value at 37 °C when the pH-dependent controlled-release is investigated; while the dialysis is carried out at a certain temperature at pH 3 or pH 5 when temperature-depended adjustable controlled-release is

studied. After mixing a known volume of microcapsule dispersion with the same volume of pure water, the permeability of VB12 across the capsule membrane is measured by determining the increase of VB12 concentration in the surrounding medium with time through an on-line monitoring UV-vis Spectrometer (UV-1700, Shimadzu) at a wavelength

of 361 nm. The permeability coef?

cient of VB12 across the capsule membrane, P VB12 , can be calculated using the following equation derived from Fick's ? rst law of diffusion: [ 11,12,37 ] ()=??P V V A V V t C C ln VB12s m s m f i f t (1)

w here C i ,C t , and C f are the initial, intermediate (at time

t ) and ? nal concentrations of VB12 in the surrounding medium respectively, V m

F igure 10. C ontrolled-release characteristics of model drug VB12 from

CS-M-T microcapsules prepared with different concentrations of temper-ature-responsive sub-microspheres in buffer solution of pH 3 at 37 °C.

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and V s are the total volume of microcapsules and the volume of the

surrounding medium respectively, and A is the total surface area of microcapsules.

T o load the FITC-dextran, the microcapsules are dialyzed against FITC-dextran aqueous solution (1 gL ?1 ) for more than three days. In the

controlled-release experiments, the microcapsules are ? rst equilibrated

in buffer solution of pH 7.4 containing FITC-dextran (1 gL ?1 ) at a certain

temperature, meanwhile, the buffer solution of pH 7.4 without FITC-dextran is also kept at the same temperature. After reaching equilibrium state, the microcapsules loaded with FITC-dextran are quickly transferred into the buffer solution of pH 7.4 without FITC-dextran. The time-dependent release behaviors of FITC-dextran from the microcapsules at test temperatures are observed and recorded by the CLSM. To quantitatively analyze the release behaviors, average ? uorescence intensity of a certain region that re? ects the release of the ? uorescent FITC-dextran is estimated by Leica Analysis Software mounted on the CLSM. The decrease of average ? uorescence intensity of a region that covers a typical microcapsule is estimated for monitoring the release behavior. The decrease of average ? uorescence intensity is characterized by the ratio of ? uorescence intensity at time t to that at the beginning. A thermostatic stage that mounted on the CLSM is used for temperature control throughout the experiments.

A cknowledgements

T

he authors gratefully acknowledge support from the National Natural Science Foundation of China (21136006, 21276002, 21322605), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1163), the Program for New Century Excellent Talents in University (NCET-11-0352), and the Foundation for the Author of National Excellent Doctoral Dissertation of China (201163).

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